A bio-molecule specifically binding to a target substance or a low molecular compound targeting a biomolecule is expected as a medicinal drug candidate, which binds specifically to a target substance, thereby producing a useful physiological activity in a living body. Such a bio-molecule or a low molecular compound is also expected as a target-substance capturing molecule of a biosensor by using the specific binding ability thereof to the aforementioned target substance.
As an example of such a bio-molecule, an antibody may be mentioned. The antibody specifically binds to various types of foreign substances invading into the body fluid of an animal by recognizing various structures on the surfaces of the foreign substances and detoxicates them by the immune system. In short, the antibody is one of the proteins functioning in a self-defense mechanism. To function such a mechanism effectively, the antibody has a molecular diversity (that is, having a number of different amino acid sequences in order to bind to various foreign substances). The number of kinds of antibodies is estimated 107 to 108 per animal individual. Since an antibody has such specific antigen recognition ability, high antigen binding ability and molecular diversity, it is expected as a medicinal drug candidate and a target-substance capturing molecule.
The antibody is a protein generally 150 kDa consisting of two of two types of polypeptide chains; one is called a heavy chain of about 50 kDa, and the other is called a light chain of about 25 kDa.
The heavy chain and the light chain each have a variable region and a constant region. The light chain is a polypeptide chain constituted of two domains; one is a variable region (called a light chain variable region: VL) and the other is a constant region (CL). On the other hand, the heavy chain is a polypeptide chain constituted of 4 domains, that is, a single variable region (heavy chain variable region: VH) and three constant regions (CH1 to CH3). Each domain consists of about 110 amino acids and has a cylindrical structure, in which β-sheets are arranged in antiparallel and mutually connected via an S—S bond to form a very stable layer structure.
Antibody molecules characteristically have the binding diversification capable of binding to various types of antigens. The binding diversification is ascribed to the diversity in amino acid sequences of three complementarity determining regions (CDRs) having a loop structure and present in each of the variable regions (VH and VL). The CDR is also called a hypervariable region. Each domain of the VH and VL has three CDRs. These CDRs are arranged on the surface of an antibody molecule and separated from each other by a region called a framework, which has a relatively common amino acid sequences between the VH and VL domains. The antibody recognizes a spatial arrangement of functional groups of a recognition site (antigenic determinant: epitope) of an object, a target substance by its CDR's configuration. By virtue of this, the antibody can recognize a molecule very specifically.
Antibodies can be produced by a method in which a desired antigenic substance is injected in combination with an adjuvant to an animal recipient (such as a rabbit, goat or mouse) at predetermined time intervals and antibodies present in the serum are recovered. Antibodies can be also produced by another method in which B cells capable of producing the antibodies are taken from the aforementioned animal recipient, fused with established tumor cells to prepare hybridoma cells, and then, the hybridoma cells are allowed to produce antibodies, followed by purifying the antibodies.
The antibodies produced by the former method contain various types of antibodies (a mixture of antibodies) recognizing different structures on the surface of the antigenic substance used in immunization. Such a serum containing a plurality of antibodies binding to a single antigen is called a polyclonal antibody. The antibodies produced by the latter method are called a monoclonal antibody. This is because since the antibody-producing B cells can produce only one type of antibody. The antibodies produced from one of the hybridoma cells mentioned above come to be single-type monoclonal antibodies.
In either method, an animal must be immunized with a target substance, an antigen. Whether an antibody, that is, a capturing molecule binding to a desired target substance, is obtained or not cannot be confirmed until the antibodies or the serum is taken and its titer (avidity) is checked. In short, in either a polyclonal antibody or a monoclonal antibody, the characteristics of the antibody obtained vary depending upon the immune system of an animal to be immunized. Furthermore, even if the hybridoma cells capable of producing a monoclonal antibody exhibiting a binding ability to a target substance can be obtained, an efficient genetic engineering method has not yet been found for improving the binding ability of the obtained antibody, at present. Moreover, generally, production of an antibody against a target substance having an analogous structure to that of a bio-constituent of an animal recipient, such as a sugar and a lipid, cannot be expected even if it is a non-self substance. In other words, production of an antibody specifically binding to such a target substance cannot be expected in the immune system serving as a bio-defense system.
On the other hand, a combinatorial method is disclosed to obtain a capturing molecule binding to a target substance by using, for example, an antibody fragment containing at least a part of VH and VL, serving as a binding portion (such as Fab and a single chain Fv (scFv)) of an antibody to an antigen. In U.S. Pat. No. 5,969,108, there is a known technique that an antibody fragment as described above is fused with a phage, in particular, a coating protein of a fibrous phage, and used as a phage antibody having an antibody exposed on the surface. Such a phage having an antibody exposed on the surface of the coating protein is disclosed in not only U.S. Pat. No. 5,969,108 but also the pamphlet of International Publication WO88/06630, WO92/15606, those disclose that the phage is used in a method of selecting a clone of an antibody fragment. According to these methods, a clone capable of binding a target substance can be easily obtained compared to a conventional immunization method for obtaining an antibody. In short, a conventional method for producing antibodies, which is said to be difficult to express other than in animal cells, can be improved by cleaving an antibody into fragments to lower the molecule weight.
In an antibody exposure method represented by the aforementioned method, first, a leading antibody fragment binding to a target substance is obtained under a specific selection pressure and mutated by a genetic engineering approach. Then, a binding/selection experiment is repeatedly performed. As a result, an antibody fragment having a higher binding ability to the target substance can be obtained. These antibody exposure methods have a characteristic feature in that since the complicated immune system of a living body is not used to obtain an antibody to be bound to a target substance, it does not a matter whether an antigen is self-derived or nonself-derived. Furthermore, if a gene portion encoding the CDR portion of an antibody fragment is chemically synthesized, the size of a gene library also can be enlarged.
J. Mol. Biol., 1995, 246, 367-373 discloses improving the binding ability to a target substance HEL. To be more specifically, the document discloses that scFv (derived from D1.3 and HyHEL10) capable of binding to HEL is genetically fused to obtain a single stranded scFv dimer, which shows improved binding ability to HEL. Similarly, International Publication WO2004/003019 pamphlet also suggests a technology regarding a target substance-capturing molecule that recognizes two different epitopes present on the surface of the same single target substance molecule although unfortunately, it fails to mention specific techniques.
However, even in the antibody and antibody molecule obtained by such a combinatorial method and genetic engineering approach, it is still difficult to obtain a clone having an excellent binding ability to a substance such as a sugar and a lipid, at present.
On the other hand, it has been suggested that the in-vivo behavior of a lipid and post-translational modification of a protein have biologically significant meanings. Therefore, it is expected to apply them to not only biochemical/medical fields but also wide variety of fields.
In recent years, attempts have been made to design and prepare a capturing molecule using an oligopeptide and a protein molecule other than an antibody or an antibody fragment. Such a candidate protein for use in a novel capturing molecule can be selected by a combinatorial method from a molecule library having molecular diversity genetically produced by taking an advantage of a stable molecular structure (J. Mol. Recognit., 2000, 13, 167-187). In most of these molecules, a β-sheet structure present in the molecules is used, more specifically, a loop structure which is considered less contributable to stabilization of the entire structure of the molecule between not less than two strands, is genetically manipulated to impart diversity. In this respect, diversification of these molecules is produced in the same manner as in an antibody. Furthermore, the mechanism for recognizing a target substance by a plurality of loops may be similar to that by an antibody. Representative examples of such a molecule include anticolin (Review in Molecular Biotechnology, 74: p 257, 2001), fibronectin and type III domain (J. Mol. Biol, 284:p 1141, 1998).
Recently, it is proposed that a molecule having an α-helix as a basic structure recognizes a target substance in a different mechanism from that of a β-sheet structure represented by an antibody (Nature Biotechnol. 15, 1998, 772-777, Nature Biotechnol, 2004, 22, 575-582, and WO0220565).
These publications disclose the results, which suggest that a molecule binds to a target substance via an amino acid residue exposed on the surface of an α-helix in contact with a solvent (also called as a solution contact surface or an exterior surface) or via an amino acid of the peptide portion connecting between the α-helices.
Furthermore, even among proteins having the same β-sheet structure as in an antibody, a molecule which binds to a target substance via a different mechanism from that involved in the loop structure of the antibody is disclosed, wherein a target substance is bound to a concave portion formed by side chains of a β-strand forming a β-sheet structure (Biochem. J., 2004, 382, 769-781).
As a more specific example, mention is made of CBM4-2 classified in a carbohydrate binding module.
The aforementioned technique suggests that a substance rarely recognized by an antibody known in the prior art can be recognized by a different molecular recognition mechanism from that of the antibody.
However, the binding ability of a substitute antibody molecule as described above to a target substance is generally low compared to that of the antibody to the antigen of an antibody-antigen complex. For example, lipocalin and fibronectin having a KD value to a target substance falling within several to several tens of nM are disclosed. A CBM as mentioned above generally has a KD value of several to several tens of μM. It is presumed that such binding abilities of these substitute antibody molecules are too low to produce a product having a desired ability sufficient to capture a target substance. When such a substitute antibody molecule is used in a sensor, an SN ratio of sensitivity, ascribed to the non-specific interaction between proteins, presumably increases. Therefore, even if a novel capturing molecule capable of capturing a substance that has a low binding ability to an antibody, the binding specificity of the novel capturing-molecule is not considered sufficient in view of application to a product. A technical problem still remains.